The present invention is directed to the field of animal and human health, and more particularly to pharmacological uses of analogs or mutants of interferon-tau (IFN-xcfx84) that differ from native IFN-xcfx84 because of substitutions of amino acids near the amino terminus of the IFN-xcfx84 molecule that impart improved biological activity. The IFN-xcfx84 mutants described in this disclosure have low toxicity, retain the same or slightly reduced antiviral activity compared with highly effective IFN-alpha, and have enhanced antiproliferative activity compared to native IFN-tau, making them useful in treating viral infections, cancer, and immune system diseases including autoimmune diseases. The present invention is also directed to a method for making novel recombinant proteins, especially interferons, interleukins, and cytokines, polypeptide hormones and other biopharmaceuticals that have improved biological activity over known proteins and/or lower toxicity and/or increased stability.
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The term xe2x80x9cinterferonxe2x80x9d generically refers to a group of vertebrate glycoproteins and proteins that are known to have various biological activities, including antiviral, antiproliferative, and immunomodulatory properties at least in the species of animal from which such substances are derived and sometimes the interferons have cross species activity. The following definition of interferon has been accepted by an international committee assembled to devise a system for the orderly nomenclature of interferons: xe2x80x9cTo qualify as an interferon a factor must be a protein which exerts virus nonspecific, antiviral activity at least in homologous cells through cellular metabolic processes involving synthesis of both RNA and protein.xe2x80x9d Journal of Interferon Research, 1, pp. vi (1980). xe2x80x9cInterferonxe2x80x9d as used to describe the present invention shall be deemed to have that definition. The type I interferons (IFN-xcex1, -xcex2, -xcfx89, and -xcfx84) are a group of proteins produced by the body to defend cells by inhibiting viral replication and decreasing cell proliferation.
IFN-alphas have been shown to inhibit various types of cellular proliferation. IFN.alpha.""s are especially useful against hematologic malignancies such as hairy-cell leukemia (Quesada, et al., 1984). Further, these proteins have also shown activity against multiple myeloma, chronic lymphocytic leukemia, low-grade lymphoma, Kaposi""s sarcoma, chronic myelogenous leukemia, renal-cell carcinoma, urinary bladder tumors and ovarian cancers (Bonnem, et al., J. Bio. Response Modifiers 3:580(1984); Oldham, Hospital Practice 20:71(1985). The role of interferons and interferon receptors in the pathogenesis of certain autoimmune and inflammatory diseases has also been investigated (Benoit, et al., J. Immunol 150(3):707(1993).
IFN-alphas are also useful against various types of viral infections (Finter, et al., Drugs 42(5):749(1991). Alpha interferons have shown activity against human papillomavirus infection, Hepatitis B, and Hepatitis C infections (Finter, et al., 1991; Kashima, et al., (Laryngoscope 98:334(1988); Dusheiko, et al. J. Hematology 3 (suppl.2):S199(1986); Davis, et al., N. England J. Med. 321:1501(1989). The journal articles listed in this application are all incorporated in their entirety.
Interferons of the alpha type (IFN-xcex1s) are FDA-approved for the treatment of several diseases, including chronic hepatitis B and C, genital warts, hairy cell leukemia, and Kaposi""s sarcoma. They are in clinical trials for AIDS and cancers, including non-Hodgkin""s lymphoma and malignant melanoma (Mariano, T. M., Interferons: Principles and Medical Applications, 1992, 129-138(1992), chronic myelogenous leukemia, cutaneous squamous cell carcinoma, and laryngeal papillomatosis (Baron, S., JAMA 10, 1375-83(1991).
IFNs are also able to aid the body by acting in an immunomodulatory role. For example, type I IFNs have been shown to increase macrophage phagocytic activity and nitrous oxide mediated killing as well as regulate IFN-xcex3 production (Reder, A., Interferon Therapy of Multiple Sclerosis, 61-64, 485-492(1997). Like other type I interferons, IFN-xcfx84 production has also been shown to increase natural killer cell activity (Tuo, W., American Journal of Reproductive Immunology, 29, 26-34(1993).
However, IFN-xcex1 therapy has its drawbacks, as patients often suffer side effects of the treatment, which may be severe. Side effects are dose-dependent, and low dose side effects include flu-like symptoms which frequently interfere with normal activity, while higher doses may induce nausea, vomiting, anorexia, and rashes (Pontzer, et al., Cancer Res. 51:5304(1991). Extremely high doses appear to cause peripheral neuropathy and thrombocytopenia. It has also been previously demonstrated that in vivo treatment with IFN-xcex2 and IFNxcex1 in humans and animals causes toxicity manifested as a number of side effects including fever, lethargy, tachycardia, weight loss, and leukopenia. These side effects often require (i) the interferon dose to be reduced to levels that limit the effectiveness of treatment, or (ii) termination of the treatment. Thus, treatment with alpha interferons is constrained both by patient compliance and the inability to use high doses in a sustained fashion. Adverse effects of IFN-beta are similar to those seen with IFN-alpha. IFN-beta has been used for treatment of multiple sclerosis.
A more recently discovered type 1 interferon, interferon-tau (IFN-xcfx84), has lower toxicity than IFN-xcex1 while also displaying antiviral and antiproliferative activities. Ovine IFN-xcfx84 is a major conceptus secretory protein produced by theembryonic trophectoderm during the critical period of maternal recognition in sheep. It is produced and secreted in large amounts for a short time prior to implantation. Its principle role in sheep and other ruminants is to prevent regression of the corpus luteum by inhibiting estrogen receptor upregulation and blocking the pulsatile secretion of prostaglandin F2 alpha .
Relative to other interferons, ovine IFN-tau. shares about 45 to 68% amino acid homology with Interferon-alpha and the greatest sequence similarity with the interferon-omega-s (IFN omega-s) of about 68%. Ovine IFN-xcfx84 has about 50% sequence homology to interferon xcex1, with the closest homology in the carboxy terminal region of the molecule. Like IFN-alpha, FN-T also has five helices. The amino acid sequence for human IFN xcex1A is provided as SEQ. ID NO. 1. The amino acid sequence for native ovine IFNxcfx84 1mod is provided as SEQ. ID NO. 2 and its nucleic acid sequence is SEQ. ID NO. 3.
There has been significant interest in finding functionally important sites on Type I IFNs, and in developing novel IFNs with improved biological activity, significant cross species activity and low toxicity for clinical use in treating immune diseases and cancer.
It is an object of the present invention to provide a method for making a recombinant protein having improved biological activity by changing one or more amino acids in a first protein with a known biological activity, which amino acids differ from corresponding amino acids in a structurally similar second protein having the desired biological activity, to the differing one or more amino acids found in the second protein, in order to produce a compound with an improved biological activity. In a preferred embodiment the differing amino acids are in an area of the molecule with a known biological activity. The recombinant proteins can be any protein, especially interferons, interleukins, cytokines, polypeptide hormones or other biopharmaceuticals. In one embodiment, the first and second proteins are type one interferons, including interferon tau, alpha and beta and omega. In another embodiment, the first protein is interferon-tau and the second protein is interferon-alpha.
It is a further object of this invention to provide recombinant proteins with enhanced antiviral and/or antiproliferative activity, or lower cytotoxicity.
In a further object of the invention, the amino acid substitutions are chosen by identifying solvent-exposed amino acids in the first and second proteins, and making amino acid substitutions in the first protein by inserting the differing solvent-exposed amino acids that were identified in the second protein. The first and second proteins in the method of the present invention may be from the same or different species, and may be naturally occurring or non-naturally occurring.
Another objective of the present invention is to provide novel interferon-tau mutant proteins with low cytotoxicity for use in treating or preventing cancer, immune system diseases including autoimmune diseases such as Type I diabetes mellitus, rheumatoid arthritis, lupus erythematosus and psoriasis; and viral infections, or for any other use that interferons may generally be put to. The novel interferon-tau mutants of the present invention are identified by amino acid SEQ. ID NOs. 4-10, and 18-20.
It is another object to provide (a) a method for treating cancer or a tumor, comprising administering to an animal in need of such treatment, a therapeutically effective amount of a recombinant interferon tau protein selected from the group comprising amino acid sequences identified by SEQ. ID NOs. 4-10, and 18-20, sufficient to treat the cancer; including human adenocarcinoma, treat breast cancer, prostate cancer, glioblastomas, melanomas, myelomas, lymphomas, leukemias, lung cancer, skin cancer, bladder cancer, kidney cancer, brain cancer, ovarian cancer, pancreatic cancer, uterine cancer, bone cancer, colorectal cancer, cervical cancer and neuroectodermal cancer, and premalignant conditions, including, without limitation, monoclonal gammapothies, dysplasia, including, without limitation, cervical and oral dysplasia. (b) a method of treating a viral disease, comprising administering to an animal in need of such treatment, a therapeutically effective amount of a recombinant interferon tau protein selected from the group comprising amino acid sequences identified by SEQ. ID NOs. 4-10, and 18-20, sufficient to treat the viral disease as enumerated in the detailed description of this invention and including RNA and DNA viruses, HIV, and Hepatitis B and C, especially; (d) a method of decreasing tumor cell growth, comprising contacting tumor cells in vivo or in vitro, with a recombinant IFN-tau protein selected from the group comprising amino acid sequences identified by SEQ. ID NOs. 4-10, and 18-20, at a concentration effective to decrease growth of the tumor cells.